1. Field of the Invention
The present invention provides hydrophilic IR-transparent microporous membranes, methods of making said membranes by treatment of hydrophobic microporous membranes with plasma, spectroscopic sample holders composed of the hydrophilic IR-transparent microporous membranes and methods of using the hydrophilic IR-transparent microporous membranes. More particularly, the present invention provides hydrophilic IR-transparent microporous membranes and methods of using said membranes to identify bacterial impurities in food using infrared spectroscopy.
2. Background
In infrared (“IR”) spectroscopy, a beam of light from an infrared source is passed through a sample. The light that is transmitted through the sample is evaluated in comparison with the incident light and its intensity plotted as a function of wavelength or wavenumber. Wavenumber is expressed herein as centimeters−1 or “cm−1”. This spectral plot or spectrum can provide information regarding the functional groups and structural features of the sample and, accordingly, IR spectroscopy has become a valuable tool in analytical chemistry for certain types of samples.
The infrared region of the electromagnetic spectrum extends from the upper end of the visible region (wavenumber of approximately 14,300 cm−1) to the microwave region (near 20 cm−1). The region which is typically of most interest to analytical chemists for determination of structural features of an organic sample is from about 4000 cm−1 to about 400 cm−1. In this region of the spectrum, organic compounds absorb incident infrared light at frequencies corresponding to the vibrational frequencies of the compound. These absorbed frequencies are characteristic of the structural features of the compound or compounds in the sample and can permit rapid identification. The intensities of the peaks in the spectral plot or spectrum are a function of the concentration of the sample, extinction coefficient, and path length of the incident light through the sample.
To obtain an infrared spectrum of a sample, the sample is typically applied to a sample holder or “cell”. This sample holder or cell holds the sample in the path of the incident beam of infrared light. It is essential that the material used for the sample holder be highly transmissive in that region of the IR spectrum which is of interest. Also, the sample holder should not be soluble in, or reactive with, either the sample or solvent (if any). Illustrative examples of materials used in sample holders include inorganic salts, glasses, and quartz.
Sodium chloride (NaCl) is perhaps the most commonly used material since it does not absorb infrared light in the range of 4000 to 625 cm−1 and is relatively less expensive than some alternatives. However, NaCl crystals are very susceptible to moisture and easily broken. For a discussion of cell materials see Pasto and Johnson, Organic Structure Determination, Prentice-Hall, Inc., 1969, pp. 145-147.
In the majority of analyses, the holder (or cell) is a pair of plates made from crystals of an inorganic salt that has been precisely machined and polished for maximum optical clarity. A sample is then placed between the pair of plates and mounted by a variety of techniques in the beam of infrared light. Solid samples are often ground and intimately mixed with an inorganic salt such as potassium bromide, pressed into a thin wafer or pellet, applied to a sample holder, and mounted in the infrared beam. Alternatively, samples may be mulled with an oil such as NUJOL™ mineral oil, applied to a sample holder, and analyzed as a thin film. Liquid samples, either neat or in solvent, may also be analyzed using a sealed cell in which a pair of plates are sealed together with a spacer to provide a chamber in which the sample is held. In addition to the use of plates, other sample preparation techniques have been developed. For instance, liquids or solutions having a relatively high surface tension such as aqueous solutions have been analyzed by suspending a thin film from a loop of wire. Also, a solution may be coated and dried to form a film, e.g., a solution may be coated on a film of polytetrafluoroethylene and dried, and the resulting thin film peeled from the polytetrafluoroethylene and analyzed.
Due to the susceptibility of many known cell materials to degradation by moisture and the long drying time necessary for preparation of some samples, analysis of aqueous samples is difficult. Increasingly stringent regulations have prompted many industries to reduce or eliminate organic solvent use and emissions, prompting the development of water-based processes and products. Illustrative examples of materials that have been used for cells for use with aqueous samples include silver bromide, calcium fluoride, and barium fluoride. Use of such materials is limited by the typically high expense, limited useful spectral ranges, burdensome maintenance, and difficult sample preparation associated with such materials. Typically, aqueous samples are analyzed using a horizontal attenuated total reflectance (“ATR”) crystal to which a sample is applied. A beam of infrared light is reflected repeatedly through the sample before being evaluated in a detector. Use of this technique is hampered by the high cost of sample holders and difficulties encountered in sample preparation and maintenance. In part due to these problems, IR spectroscopy has not reached its potential as a routine tool for analysis of aqueous samples.
In addition to the problems described, namely cost, sensitivity to moisture and fragility, commercially available cells have high maintenance requirements. In view of the high costs, disposal of these cells is prohibitive. Accordingly, sample holders must be carefully cleaned, typically with organic solvents, after each analysis to prevent contamination from one sample to the next. In some instances, the solvents may present health risks to operators. In addition, the high cost of sample holders tends to inhibit retention of samples on a long term basis.
Dove and Hallett, Chemistry and Industry, 1966, pp. 2051-53, describe an all-plastic evacuable cell to be used for infrared or ultraviolet spectroscopic analysis of gases. The cell has windows that can be made from RIGIDEX™ Type 35 polyethylene. The relative thickness of the windows, i.e., about 3 millimeters, would preclude the use of such sample holders in most routine IR spectroscopic analysis due to the strong absorbances. Andrede, J. Chem. Ed., 66(10), p. 865, 1989, describes using polyethylene film as windows in a sample cell. For sampling of liquids the author suggests applying the sample to a film stretched over a ring, covering the sample with a second film, and securing both stretched films with a second ring.
Gagnon, U.S. Pat. No. 5,470,757, describes hydrophobic microporous polymeric membranes and the use of same as sample holders for IR spectroscopy. More particularly, Gagnon teaches that solid (e.g., in powder form), liquid, and solution samples can be supported by hydrophobic microporous polymeric membranes such that the IR spectrum of the sample could be determined. The hydrophobic microporous polymeric membranes are easy and inexpensive to use. Unfortunately, these membranes are not well suited for use with aqueous samples due to the hydrophobicity of the membranes.
Enterobacter sakazakii is a Gram-negative rod shaped bacterium that has been associated with neonate deaths and outbreaks of a rare form of infant meningitis and other diseases (Med Baltimore (2001) 80 (1): 113-122). Reported case-mortality rates were high and ranged from 40-80% among immunocompromised infants (J Food Protection (1997) 60 (3):226-230). Although the mode of transmission of this organism has not been identified, the presence of E. sakazakii in powdered infant formula milk (IFM) has been of particular concern (J Am Med Assoc (2001) 287:2204-2205). Literature surveys indicate that the incidence of E. sakazakii in IFM commercial products is low, namely 20 out of 141, 8 out of 120 (J Food Protection (1997) 60 (3):226-230), and 2 out of 82 IFM (Iverson and Forsythe 2004), and its isolation levels range from 0.36 to 66.0 cells/100 g (J Clin Microbiol (1998) 26 (4):743-6).
Established conventional procedures (Food Microbiol (2004) 21:771-777; J Clin Microbiol (1998) 26 (4):743-6; and J Food Protection (1997) 60 (3):226-230) for the isolation of E. sakazakii from dehydrated powdered infant formula include the 2002 FDA culture method which requires seven days. The FDA culture method entails pre-enrichment in distilled water, enrichment in Enterobacteriaceae enrichment broth, plating on violet red bile glucose agar, selecting five Enterobacteriaceae colonies and incubating on trypticase (tryptic) soy agar plates at 25° C. for 48-72 h to observe the yellow-pigmented colonies that are characteristic of E. sakazakii, and confirmation using the API 20E (bioMerieux, Inc, Hazelwood, Mo.) biochemical profiling system.
It would be desirable to develop faster, less expensive methods of identifying microorganism contaminants present in food samples. More particularly, it would be desirable to provide faster, less expensive methods of direct identification of harmful microorganism contaminants present in powdered foods including those contaminants present in powdered baby formula.